Amino acids represent the source of life and make up twenty percent of the human body. They are divided into two categories—essential amino acids, which are not synthesized in the body and must be taken from food; and non-essential amino acids. Phenylalanine is one of the eight essential amino acids that is an important precursor for the synthesis of tyrosine that serves as a precursor for synthesis of many neurotransmitters and thyroid hormones. Physiologic requirements for phenylalanine are met exclusively by dietary protein intake. Usual dietary intake of protein provides excess amounts of phenylalanine and blood phenylalanine levels are maintained within non-toxic levels via utilization, metabolism and excretion. However when the body is unable to metabolize phenylalanine to tyrosine, the level of phenylalanine in the body is elevated leading to a rare condition called hyperphenylalaninemia that severely impairs functions of the central nervous system.
Hyperphenylalaninemia (HPA) is a congenital metabolic disorder inherited as an autosomal recessive trait and characterized by the presence of blood phenylalanine levels that exceed the limits of the upper reference range of 2 mg/dL or 120 mmol/L. HPA is divided into (i) HPA caused due to deficiency in enzyme phenylalanine hydroxylase (PAH) that is required for the conversion of ingested phenylalanine to tyrosine, due to absent or mutated PAH enzyme; the condition being known as Phenylketonuria (PKU) or (ii) HPA resulting from a deficiency in tetrahydrobiopterin (BH4) cofactor of the enzyme PAH, due to defects in its biosynthesis or recycling.
Tetrahydrobiopterin is a biogenic amine of the naturally occurring pterin family that is a cofactor for a number of different enzymes, including phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase and nitric oxide synthase regulating their activity and catalysis. These enzymes further are rate limiting in the biosynthesis of the neurotransmitters serotonin (5-hydroxytryptamine), melatonin, dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), and nitric oxide (NO).
In order to control hyperphenylalaninemia caused due to both the conditions mentioned herewith above, dietary intervention is followed. Such dietary intervention typically demand administering to the patient, food that is natural and free from or low in phenylalanine. However such a dietary regimen, apart from providing low phenylalanine, eliminates many other sources of other essential amino acids, vitamins and minerals. Consequently such a diet provides inadequate protein, vitamins and minerals thereby hindering normal growth and development. Apart from adults, for babies too infant formulae which have low phenylalanine content are the primary food source. The phenylalanine-free protein formulae that are available are mostly bitter tasting making the food unpalatable. Further the strict regimen of dietary protein is practically impossible for patients of all ages to adhere to in daily life.
Thus there remains a need to obviate the dietary restrictions and replace or supplement the same with oral treatment by providing an oral composition of tetrahydrobiopterin.
Further BH4-responsive PAH deficiency has also been diagnosed as a variant of hyperphenylalaninemia or phenylketonuria caused by mutations in the human PAH gene that responds to oral BH4 loading by stimulating enzyme activity and therefore lowering serum phenylalanine. BH4 is said to have a chaperon-like effect on PAH synthesis and/or is a protecting cofactor against enzyme auto-inactivation and degradation.
Therefore, since administration of BH4, alone has been proven effective in the treatment of BH4-responsive hyperphenylalaninemia, for which the only available treatment has been a diet therapy, development of effective treatments for the disease, in particular, development of effective tetrahydrobiopterin preparations is an urgent need.
Sapropterin dihydrochloride is a synthetic version of naturally occurring tetrahydrobiopterin. Sapropterin dihydrochloride is chemically represented as (6R)-2-amino-6-[(1R,2S)-1,2-dihydroxypropyl]-5,6,7,8-tetrahydro-4(1H)-pteridinone dihydrochloride. The 6R-form is pharmacologically effective while the 6S form may cause inactivation of phenylalanine hydroxylase, thus inhibiting the effects of the 6R form. Sapropterin dihydrochloride is a crystalline powder, hygroscopic and very soluble in water with solubility being greater than 1 g/ml. It exhibits polymorphism and many crystalline forms have been identified; among all the polymorphic forms, Form B was identified to be thermodynamically stable crystalline anhydrate form. Sapropterin dihydrochloride is currently available as oral soluble tablets of 100 mg under the brand name Kuvan™. It is marketed by BioMarin in the US and Merck Serono in Europe. Kuvan™ has been designated as an orphan medication since hyperphenylalaninemia is a rare disease. Kuvan™ is indicated to reduce blood phenylalanine levels in patients with hyperphenylalaninemia due to tetrahydrobiopterin responsive phenylketonuria. It is to be used in conjunction with phenylalanine restricted diet. In patients with phenylketonuria the role of sapropterin dihydrochloride is to enable endogenous phenylalanine hydroxylase activity and to partially restore oxidative metabolism of phenylalanine, resulting in decreased blood phenylalanine levels. In patients with BH4 deficiency, sapropterin dihydrochloride is proposed to restore endogenous phenylalanine hydroxylase activity by providing an exogenous source of the missing cofactor.
Tetrahydrobiopterin is an unstable compound; at ambient temperature it is prone to autoxidation in the presence of molecular oxygen (Davis et al., Eur. J. Biochem., Vol 173, 345-351, 1988). It also undergoes auto-oxidation in aqueous solutions at pH 7.4 to form 7,8-dihydrobiopterin (BH2) (Thöny et al., 2000). Tetrahydrobiopterin is also very hygroscopic. Therefore the development of stable oral composition comprising tetrahydrobiopterin that is prone to degradation at room temperature is a challenging task.
The formulation of Kuvan™ as disclosed in the U.S. Pat. No. 7,566,462 describes use of polymorph B, of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride, an antioxidant, and a pharmaceutically acceptable excipient, diluent, or carrier for preparation of stable tablet formulation; wherein a specific weight ratio of the antioxidant to the (6R)-L-erythro-tetrahydrobiopterin dihydrochloride of about 1:5 to about 1:30 has been used. Such a composition after six months in a container at room temperature and about 60% humidity is said to retain at least about 95% of the initial amount of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride. Kuvan™ has a shelf life of 3 years when stored below 25° C. Further European Publication 1757293A1 discloses a pharmaceutical preparation for the treatment of BH4-responsive hyperphenylalaninemia provided in the form of granule, fine granule, or dry syrup, comprising sapropterin hydrochloride as an active ingredient; a flavoring agent; a coloring agent which is stable to acid and oxidation; and ascorbic acid or L-cysteine hydrochloride as a stabilizer, wherein the preparation has a moisture content (weight loss on drying) of 0.9% or less. This European Publication 1757293A1 discloses that the decomposition of sapropterin hydrochloride caused by moisture can be prevented by keeping the moisture content of the preparation at 0.9% or lower during the production. U.S. Pat. No. 4,778,794 discloses pharmaceutical compositions comprising in addition to carriers, antioxidants that stabilize tetrahydrobiopterin; with the weight ratio of the antioxidant to active ranging from 0.2-1.5. Further tetrahydrobiopterin tablets from Schircks Laboratories contain antioxidant ascorbic acid in a ratio of 1:1 with active and at room temperature these tablets have a shelf life of 2 months and at 5° C. or colder are stable for 4 months.
Thus though researchers have developed compositions of sapropterin comprising stabilizers in variety of ratios, the stability of these compositions is low at room temperature or 40° C./75% relative humidity and need to be stored under refrigeration. Low stability of such tetrahydrobiopterin compositions is commercially undesirable and significant degradation due to improper storage could hinder therapy. Need therefore, exists for preparations of tetrahydrobiopterin that are more stable and retain desired amount of active over a longer time even when not refrigerated.
Further the amount and type of stabilizer and other excipients present in the compositions of sapropterin determine the stability of the active and compositions thereof. Too little or too much stabilizer can affect the stability of the compositions of sapropterin and an appropriate amount of stabilizer must therefore be present in these compositions. Further tetrahydrobiopterin also decomposes in the presence of moisture and it may also react with reducing sugars or may cause discoloration of some excipients due to its strong reducing power. The stability of sapropterin also needs to be ensured during the process of preparation of compositions thereof.
Need therefore exists to develop stable compositions of tetrahydrobiopterin that have adequate amount of stabilizing agents and/or other excipients therein. The present inventors after thorough research have overcome the challenges associated with stabilization of tetrahydrobiopterin and developed oral composition comprising tetrahydrobiopterin and at least one stabilizing agent that are stable over a longer period of time even when not stored under refrigeration. The stable compositions of tetrahydrobiopterin according to the present invention thus provide desired amount of active over the entire shelf life of the product.